The invention is in the field of genetic analysis (genotyping).
The ability to detect variations in nucleic acid sequences is of great importance in the field of medical genetics: the detection of genetic variation is essential, inter alia, for identifying polymorphisms for genetic studies, to determine the molecular basis of inherited diseases, to provide carrier and prenatal diagnosis for genetic counseling and to facilitate individualized medicine. Detection and analysis of genetic variation at the DNA level has been performed by karyotyping, analysis of restriction fragment length polymorphisms (RFLPs) or variable nucleotide type polymorphisms (VNTRs), and more recently, analysis of single nucleotide polymorphisms (SNPs), see e.g. Lai E, et al., Genomics, 1998, 15;54(1):31-8; Gu Z, et al., Hum Mutat.1998;12(4):221-5; Taillon-Miller P, et al., Genome Res. 1998;8(7):748-54; Weiss K M., Genome Res. 1998;8(7):691-7; Zhao L P, et al., Am J Hum Genet. 1998; 63(1):225-40.
A wide variety of techniques have been developed for SNP detection and analysis, see, e.g. Sapolsky et al. (1999) U.S. Pat. No. 5,858,659; Shuber (1997) U.S. Pat. No. 5,633,134; Dahlberg (1998) U.S. Pat. No. 5,719,028; Murigneux (1998) WO98/30717; Shuber (1997) WO97/10366; Murphy et al. (1998) WO98/44157; Lander et al. (1998) WO98/20165; Goelet et al. (1995) WO95/12607 and Cronin et al. (1998) WO98/30883. In addition, ligase based methods are described by Barany et al. (1997) WO97/31256 and Chen et al. Genome Res. 1998;8(5):549-56; mass-spectroscopy-based methods by Monforte (1998) WO98/12355, Turano et al. (1998) WO98/14616 and Ross et al. (1997) Anal Chem. 15, 4197-202; PCR-based methods by Hauser, et al. (1998) Plant J. 16,117-25; exonuclease-based methods by Mundy U.S. Pat. No. 4,656,127; dideoxynucleotide-based methods by Cohen et al. WO91/02087; Genetic Bit Analysis or GBA(trademark) by Goelet et al. WO92/15712; Oligonucleotide Ligation Assays or OLAs by Landegren et al.(1988) Science 241:1077-1080 and Nickerson et al.(1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927; and primer-guided nucleotide incorporation procedures by Prezant et al.(1992) Hum. Mutat. 1:159-164; Ugozzoli et al.(1992) GATA 9:107-112; Nyrxc3xa9en et al. (1993) Anal. Biochem. 208:171-175.
The invention provides methods, compositions and systems for detecting multiple SNPs in a population of target polynucleotides in parallel. In general, the methods comprise the steps of: (a) combining target polynucleotides, capture polynucleotides and SNP probes under conditions wherein: each target polynucleotide comprises a different capture region and a different SNP region comprising a corresponding different SNP, the capture polynucleotides are immobilized and arrayed at corresponding discrete elements on a substrate and each capture polynucleotide comprises a sequence which specifically hybridizes to a corresponding different capture region, each SNP probe comprises a sequence complementary to a corresponding SNP region, the target polynucleotides are immobilized by hybridizing to the capture polynucleotides whereby each target polynucleotide is immobilized at a corresponding discrete element of the substrate, and the relative affinity of each SNP probe for the corresponding SNP region is sufficient to provide selective hybridization of the SNP probe to the corresponding SNP region, whereby each SNP probe hybridizes to the corresponding SNP region; and (b) detecting the presence of each SNP probe on the substrate, wherein the presence of a given SNP probe at a given element indicates the presence of the corresponding SNP in the corresponding target polynucleotide.
In a particular embodiment, the relative affinity of each SNP probe for the corresponding SNP region is increased sufficiently to provide enhanced selective hybridization of the SNP probe to the corresponding SNP region, whereby each SNP probe hybridizes to the corresponding SNP region; and (b) detecting the presence of each SNP probe on the substrate, wherein the presence of a given SNP probe at a given element indicates the presence of the corresponding SNP in the corresponding target polynucleotide.
In particular embodiments, the SNP probes are a subset of assay probes and the relative affinity of each SNP probe for the corresponding SNP region is increased at least in part by at least one of: (a) a method comprising the step of including in the combining step a reagent which normalizes the melting temperatures of the assay probes and (b) the SNP probe interacting with the capture polynucleotide or an auxiliary probe hybridized to the corresponding target polynucleotide, e.g. through a minor groove binder tethered to at least one of the SNP probe, the capture polynucleotide and the auxiliary probe.
In other embodiments of the general method, the assay probes comprise a degenerate set of all possible same-sized polynucleotides; the target polynucleotides are a subset of sample polynucleotides and the sample polynucleotides comprise fragmented genomic DNA; the capture polynucleotides are a subset of immobilized polynucleotides and the immobilized polynucleotides are a segregated and arrayed cDNA library; the capture polynucleotides are immobilized and arrayed at corresponding discrete elements in high density; and/or each SNP probe comprises a label.
The subject compositions include reagent sets and/or mixtures suited to the disclosed methods and the subject systems may further comprise kits and/or equipment suited to the disclosed methods.